Bacillus thuringiensis (hereinafter "B.t.") is a gram-positive soil bacterium that produces crystal proteins during sporulation which are specifically toxic to certain orders and species of insects. Many different strains of B.t. have been shown to produce insecticidal crystal proteins. Compositions including B.t. strains which produce insecticidal proteins have been commercially available and used as environmentally acceptable insecticides because they are quite toxic to the specific target insect, but are harmless to plants and other non-targeted organisms.
A number of genes encoding crystal proteins have been cloned from several strains of B.t. A review of such genes is set forth in H. Hofte et al., Microbiol. Rev., 53, pp.242-255 (1989). This reference provides a good overview of the genes and proteins obtained from B.t. and their uses, adopts a nomenclature and classification scheme for B.t. genes and proteins, and has an extensive bibliography.
The B.t. crystal protein is toxic in the insect only after ingestion. After ingestion, the alkaline pH and proteolytic enzymes in the insect mid-gut solubilize the crystal allowing the release of the toxic components. These toxic components disrupt the mid-gut cells causing the insect to cease feeding and, eventually, to die. In fact, B.t. has proven to be an effective and environmentally safe insecticide in dealing with various insect pests.
As noted by Hofte et al., the majority of insecticidal B.t. strains are active against insects of the order Lepidoptera, i.e., caterpillar insects. Other B.t. strains are insecticidally active against insects of the order Diptera, i.e., flies and mosquitoes, or against both lepidopteran and dipteran insects. In recent years, a few B.t. strains have been reported as producing crystal protein that is toxic to insects of the order Coleoptera, i.e., beetles.
The first isolation of a coleopteran-toxic B.t. strain is reported by A. Krieg et al., in Z.angew. Ent., 96, pp.500-508 (1983); see also A. Krieg et al., Anz. Schaedlingskde., Pflanzenschutz, Umweltschutz, 57, pp.145-150 (1984) and U.S. Pat. No. 4,766,203, issued Aug. 23, 1988 of A. Krieg et al. The strain, designated B.t. var. tenebrionis, is reported to be toxic to larvae of the coleopteran insects Agelastica alni (blue alder leaf beetle) and Leptinotarsa decemlineata (Colorado potato beetle). B.t. tenebrionis makes an insecticidal crystal protein reported to be about 65-70 kilodaltons (kDa) (U.S. Pat. No. 4,766,203; see also K. Bernhard, FEMS Microbiol.Lett., 33, pp.261-265 (1986)).
V. Sekar et al., Proc. Natl. Acad. Sci. USA, 84, pp.7036-7040 (1987), report the cloning and characterization of the gene for the coleopteran-toxic crystal protein of B.t. tenebrionis. The size of the protein, as deduced from the sequence of the gene, was 73 kDa, but the isolated protein contained primarily a 65 kDa component. Hofte et al., Nucleic Acids Res., 15, p.7183 (1987), also report the DNA sequence for the cloned gene from B.t. tenebrionis, and the sequence of the gene is identical to that reported by Sekar et al. (1987).
McPherson et al., Bio/Technology, 6, pp.61-66 (1988), disclose the DNA sequence for the cloned insect control gene from B.t. tenebrionis, and the sequence is identical to that reported by Sekar et al. (1987). E. coli cells and Pseudomonas fluorescens cells harboring the cloned gene were found to be toxic to Colorado potato beetle larvae.
PCT International Publication No. WO 91/07481 dated May 30, 1991, of Novo Nordisk A/S, describes B.t. mutants that produce high yields of the same insecticidal proteins originally made by the parent strains at lesser yields. Mutants of the coleopteran-toxic B.t. tenebrionis strain are disclosed.
A coleopteran-toxic strain, designated B.t. var. san diego, is reported by C. Herrnstadt et al., Bio/Technology, 4, pp.305-308 (1986), to produce a 64 kDa crystal protein that was toxic to various coleopteran insects: strong toxicity to Pyrrhalta luteola (elm leaf beetle); moderate toxicity to Anthonomus grandis (boll weevil), Leptinotarsa decemlineata (Colorado potato beetle), Otiorhynchus sulcatus (black vine weevil), Tenebrio molitor (yellow mealworm) and Haltica tombacina; and weak toxicity to Diabrotica undecimpunctata undecimpunctata (western spotted cucumber beetle).
The DNA sequence of the cloned coleopteran toxin gene of B.t. san diego is reported in C. Herrnstadt et al., Gene, 57, pp.37-46 (1987); see also U.S. Pat. No. 4,771,131, issued Sep. 13, 1988, of Herrnstadt et al. The sequence of the toxin gene of B.t. san diego is identical to that reported by Sekar et al. (1987) for the cloned coleopteran toxin gene of B.t. tenebrionis.
A. Krieg et al., J.Appl.Ent., 104, pp.417-424 (1987), report that the strain B.t. san diego is identical to the B.t. tenebrionis strain, based on various diagnostic tests.
Another new B.t. strain, designated EG2158, is reported by W. P. Donovan et al., in Mol.Gen.Genet., 214, pp.365-372 (1988) and in U.S. Pat. No. 5,024,837 issued Jun. 18, 1991, to produce a 73 kDa crystal protein that is insecticidal to coleopteran insects. The toxin-encoding gene from B.t. strain EG2158 was cloned and sequenced, and its sequence is identical to that reported by Sekar et al. (1987) for the cloned B.t. tenebrionis coleopteran toxin gene. This coleopteran toxin gene is referred to as the cryIIIA gene by Hofte et al., Microbiol.Rev., 53, pp.242-255 (1989).
The Donovan et al. '837 U.S. patent noted above also describes hybrid B.t. var. kurstaki strains designated EG2424 and EG2421, which are active against both lepidopteran insects and coleopteran insects. The beetle activity of these hybrid strains results from the coleopteran toxin plasmid transferred from B.t. strain EG2158 by conjugal plasmid transfer.
U.S. Pat. No. 4,797,279, issued Jan. 10, 1989, of D. Karamata et al. (corresponding to EP-A-0 221 024), discloses a hybrid B.t. microorganism containing a plasmid from B.t. var. kurstaki with a lepidopteran toxin gene and a plasmid from B.t. tenebrionis with a coleopteran toxin gene. The hybrid B.t. produces crystal proteins characteristic of those made by B.t. kurstaki, as well as those of B.t. tenebrionis.
U.S. Pat. No. 4,910,016, issued Mar. 20, 1990, of Gaertner et al. (corresponding to EP-A-0 303 379), discloses a novel B.t. isolate identified as B.t. MT 104 which has insecticidal activity against two orders of insects, Colorado potato beetle (Coleoptera) and cabbage looper (Lepidoptera).
European Patent Application Publication No. 0 318 143, published May 31, 1989, of Lubrizol Genetics, Inc., discloses the cloning, characterization and selective expression of the intact partially modified gene from B.t. tenebrionis, and the transfer of the cloned gene into a host microorganism rendering the microorganism able to produce a protein having toxicity to coleopteran insects. Insect bioassay data for B.t. san diego reproduced from Herrnstadt et al., Bio/Technology, 4, pp.305-308 (1986) discussed above, is summarized. The summary also includes data for B.t. tenebrionis from another source; B.t. tenebrionis is reported to exhibit strong toxicity to Colorado potato beetle, moderate toxicity to western corn rootworm (Diabrotica virgifera) and weak toxicity to southern corn rootworm (Diabrotica undecimpunctata).
European Patent Application Publication No. 0 324 254, published Jul. 19, 1989, of Imperial Chemical Industries PLC, discloses a novel B.t. strain identified as A30 which has insecticidal activity against coleopteran insects, including Colorado potato beetle larvae, corn rootworm larvae and boll weevils.
U.S. Pat. No. 4,999,192, issued Mar. 12, 1991, of Payne et al. (corresponding to EP A-0 328 383), discloses a novel B.t. microorganism identified as B.t. PS40D1 which has insecticidal activity against Colorado potato beetle larvae. B.t. strain PS40D1 is identified via serotyping as being serovar 8a 8b, morrisoni.
U.S. Pat. No. 5,006,336, issued Apr. 9, 1991, of Payne et al. (corresponding to EP-A-0 346 114), discloses a novel B.t. isolate designated as PS122D3, which is serotyped as serovar 8a8b, morrisoni and which exhibits insecticidal activity against Colorado potato beetle larvae.
U.S. Pat. No. 4,966,765, issued Oct. 30, 1990, of Payne et al. (corresponding to EP-A-0 330 342), discloses a novel B.t. microorganism identified as B.t. PS86B1 which has insecticidal activity against the Colorado potato beetle. B.t. strain PS86B1 is identified via serotyping as being serovar tolworthi.
The nucleotide sequence of a cryIIIB gene and its encoded coleopteran-toxic protein is reported by Sick et al., in Nucleic Acids Res., 18, p.1305 (1990) but the B.t. source strain is identified only via serotyping as being subspecies tolworthi. U.S. Pat. No. 4,966,155, issued Feb. 26, 1991, of Sick et al. (corresponding to EP-A-0 337 604), discloses a B.t. toxin gene obtained from the coleopteran-active B.t. strain 43F, and the gene sequence appears identical to the cryIIIB gene. B.t. strain 43F is reported as being active against Colorado potato beetle and Leptinotarsa texana.
European Patent Application No. 0 382 990, published Aug. 22, 1990, of Plant Genetic Systems N.V., discloses two novel B.t. strains bt PG51208 and bt PG51245 producing respective crystal proteins of 74 and 129 kDa that exhibit insecticidal activity against Colorado potato beetle larvae. The DNA sequence reported for toxin gene producing the 74 kDa protein appears to be related to that of the cryIIIB gene of Sick et al.
PCT International Publication No. WO 90/13651, published Nov. 15, 1990, of Imperial Chemical Industries PLC, discloses novel B.t. strains which contain a toxin gene encoding an 81 kDa protein that is stated to be toxic not only to lepidopteran insects but also to coleopteran insects, including Diabrotica.
U.S. Pat. No. 5,055,293, issued Oct. 8, 1991, of Aronson et al., discloses the use of B. laterosporous for corn rootworm (Diabrotica) insect control.
The various B.t. strains described in aforementioned literature are reported to have crystal proteins insecticidally active against coleopteran insects, but none has been demonstrated to have significant, quantifiable toxicity to the larvae and adults of the insect genus Diabrotica (corn rootworm), which includes the western corn rootworm (Diabrotica virgifera virgifera), the southern corn rootworm (Diabrotica undecimpunctata howardi) and the northern corn rootworm (Diabrotica barberi).
The B.t. strain of the present invention contains a novel toxin gene that expresses protein toxin having quantifiable insecticidal activity against the Diabrotica insects, among other coleopteran insects.